While DNA originates as the cellular mechanism for long-term genetic information storage, its highly programmable nature and predictable secondary structure folding make it an excellent material for creating nanostructures. Using DNA as a building material was first suggested in 1982 (Seeman, 1982), and in the decades since, an entire field has emerged. Duplexed double-stranded DNA (dsDNA) forms a rod, that is, remarkably rigid at length scales shorter than its persistence length (L_p ∼ 50 nm) (Mitchell et al., 2017), while single-stranded DNA (ssDNA) is very flexible (L_p ∼ 2 nm) (Chi et al., 2013; Roth et al., 2018). This enables rigid dsDNA and flexible ssDNA segments to be woven together to form pre-designed 2D sheets and 3D structures known as DNA origami (Rothemund, 2006; Douglas et al., 2009). Additionally, DNA origami is not limited to a singular static state. Various control mechanisms can be employed to dynamically switch between different engineered conformations—for instance, opening and closing a box (Andersen et al., 2009). While DNA has become a versatile building material for nanostructures and nanomachines, construction architectures using hundreds of short ssDNA strands suffer from several drawbacks that limit its scalability.

Since Rothemund’s 2006 paper introducing the concept, DNA origami has predominantly been built using their scaffold and staples architecture (Rothemund, 2006). A long ssDNA runs throughout the structure, acting as the scaffold, while hundreds of short ssDNA hybridize and staple the scaffold into its designed shape (Figure 1A). Less extensively utilized schemes composed exclusively of short oligonucleotides (Figure 1B) have also been used to create nanostructures (Ke et al., 2012; Ong et al., 2017) such as hollow rods of various circumferences (Yin et al., 2008) and the alphabet (Wei et al., 2012). These multi-stranded DNA origami (msOrigami) architectures make excellent use of DNA’s innate programmability. Over the years, the field has seen significant development, from design software (Benson et al., 2015; Veneziano et al., 2016), to curved 3D and wireframe nanostructures (Ke et al., 2009; Han et al., 2011; 2013; Zhang et al., 2015; Wang et al., 2019a). Nevertheless, msOrigami’s main drawbacks result from its multi-stranded nature: it is difficult to effectively combine reagent strands to generate high product yield, the resulting structure has multiple free ends which make it vulnerable to enzymatic degradation in biological environments, and scaling its production to industrial levels remains cost-prohibitive. These problems can be solved by simplifying the design to a single ingredient, where intramolecular interactions within a single ssDNA fold itself into a complete nanostructure (Figure 1C), like a protein.

This review discusses available intramolecular DNA secondary structures and how they are used to construct ssDNA origami (ssOrigami). SsOrigami’s benefits are highlighted: the removal of concentration dependent folding, the improved in vivo stability of ssOrigami compared to msOrigami systems, and the reduced manufacturing cost at an industrial scale. An overview of existing proof-of-concept experiments that have taken advantage of these benefits to demonstrate ssOrigami’s potential is provided.

DNA secondary structures come in two flavours—Watson-Crick (WC) base pairing (Figures 1D–G), and non-WC base interactions (nWC) (Figures 2A–F). Several excellent reviews have focused on the characterization of each structure, including paranemic cohesion (Wang et al., 2019b), the G-quadruplex (Mukundan and Phan, 2013; Cheng et al., 2021), the i-motif (Abou Assi et al., 2018), and how they work in concert to form functional DNA origami and nanodevices (Rothemund, 2006; Saccà and Niemeyer, 2012; Tian et al., 2022). While these structures were typically demonstrated in systems with intermolecular interactions, they can form just as easily, if not more, intramolecularly in ssOrigami due to an effective increased local concentration and improved stoichiometric control.

While dynamic secondary structures enable nanomachines, static secondary structures form the foundation of DNA origami’s structural components. After the development of the scaffold-staple system in 2006 (Rothemund, 2006), ssOrigami saw limited development for over a decade as research effort was poured into msOrigami. A 286-nt ssDNA tetrahedron from 2009 is one of the few constructions of this era (Li et al., 2009) alongside a 198-nt prism of ssDNA that probed the lower limits of 3D DNA origami size (He et al., 2013). As the field of msOrigami grew rapidly (Dey et al., 2021), ssOrigami at the kilobase scale remained elusive. These larger ssDNA nanostructures were challenging to synthesize due to the kinetic constraints of knotting: a singular strand threading back through a loop of itself is entropically disfavoured. Sequence design guidelines were developed in 2016 to minimize these energetic barriers and enable the design of spontaneous knot formation (Kočar et al., 2016). The most important of these is the ‘free-end’ rule which states that free ends are required at each consecutive folding step to form the correct structure. Should folding occur in the wrong order and the free end hybridizes too early, the free end is lost, and it becomes impossible to thread through the pre-folded structure. These rules were co-developed with a knotted 286-nt pyramid-shaped wireframe ssDNA nanostructure that showed proof-of-concept (Kočar et al., 2016).

A major breakthrough came in 2017 when a multi-kilobase ssOrigami was developed by avoiding topological entanglements entirely (Han et al., 2017). This new approach used paranemic cohesion coupled with single helical turns between each crossing (Figure 2G) to create structures as large as 10 kilobases (Han et al., 2017). The ssDNA sequence was designed to first form a partially paired filament driven by base pairing in the duplexed portions. Subsequently, paranemic cohesions drive the unfolded structure to fold in half at a central hinge as these bases hybridize, completing the nanostructure (Figure 2H). One half of the completed nanostructure lies on top (blue in Figure 2H) of the other half (black in Figure 2H). Not only were a wide variety of raster-filled shapes and sizes demonstrated to fold in high yield (74%–97% depending on the structure), but they were also capable of functionalization with various handles like biotin.

Combining the rules for ssDNA knotting with the paranemic cohesion of the knot-free assembly enabled the construction of knotted multi-kilobase ssOrigami (Qi et al., 2018). This design used a repeating motif of a helical domain followed by a paranemic cohesion. The ssDNA sequence first folded into a dsDNA filament with alternating paired (helical domain) and unpaired (paranemic cohesion) segments (Figure 2G). This partially paired filament then formed the designed paranemic cohesions between the unpaired regions, resulting in the fully paired, knotted ssOrigami. Following the free end rule, the paranemic cohesions were carefully engineered to fold sequentially based on melting temperature. The wrapping of strands around each other results in a knotted structure because the filament must thread back through itself in order for all of the paranemic cohesions to form (Figure 2I). Therefore, this knotting requires a wire-frame structure for free ends to pass through.

A variety of planar and 3D knotted structures with 9–57 paranemic cohesions (each driving a separate threading step) were designed and folded successfully (Qi et al., 2018). Nevertheless, this topologically threaded design is not without limitations. The major drawback is that each threading step in the structure has a misfolding rate of 4%–10% because of the entropic constraints of self-threading. This scales exponentially with the number of threading steps, so more topologically complex designs were found to have lower yield (Qi et al., 2018). A summary of existing ssOrigami nanostructures is provided in Table 1. Without additional breakthroughs enabling higher step-yields of threading, future ssOrigami designs will likely utilize predominantly unknotted structures but may introduce some threaded knots to access a more diverse range of 3D structures.

While the benefits of using ssDNA in designing static nanostructures are being addressed, the realm of dynamic ssOrigami designed using these principles is largely unexplored. The benefits of using responsive secondary structures in msOrigami design have been partially exploited (Daljit Singh et al., 2018), though many dynamic DNA nanostructures rely instead on the introduction of external oligos to induce conformational response (Andersen et al., 2009; Wickham et al., 2012; Kuzyk et al., 2014; Marras et al., 2015). Thus far, environmentally responsive nanostructures have been limited to msOrigami, but the same ideas could apply to ssOrigami. Structural and dynamic secondary structures could be combined into a single, unified sequence, preserving the advantages of ssOrigami.

SsOrigami is an underutilized and underexplored subfield in the world of DNA nanostructures. This seemingly simple design choice tempers several of drawbacks of more traditional msOrigami. Its ability to fold even at low concentration, its comparative stability in the presence of exonucleases, its production using cellular machinery, and its industrial cost-effectiveness all make ssOrigami an alternative worth pursuing. Preliminary applications of ssDNA origami in biological conditions taking advantage of these benefits have been performed, such as the sorting of liposomes (Jia et al., 2021). While existing applications are limited, in vivo biosensing and bioimaging are promising directions for ssOrigami. Additionally, with the incorporation of conditionally stable DNA interactions within the sequence, responsive ssOrigami can be designed. Benefits aside, the design limitations imposed by folding order and knotting are non-trivial barriers to entry—but these can be mitigated by clever usage of secondary structures, thermodynamic control, and topology. Additionally, benefits from DNA-based ssOrigami are shared with RNA-based ssOrigami, which has also seen recent development (Geary et al., 2014; 2021; Han et al., 2017). RNA ssOrigami has the added benefit of being genetically encoded, transcribed, and self-assembled in the same cell (Geary et al., 2014). For in vivo applications the origami can thus be synthesized within its target cell, eliminating the need for external nanostructure production and subsequent delivery into the cell—steps which traditional msOrigami would require. This has already been applied in yeast where an RNA ssOrigami nanostructure transcribed and folded in vivo was used to modulate gene expression in that same cell (Pothoulakis et al., 2022). Furthermore, the development of software specific to designing RNA ssOrigami makes entry into this subfield more accessible (Geary et al., 2021). While multi-stranded systems will continue to drive many applications in DNA origami research, single-stranded origami provides a refreshing approach for the mass production and in vivo applications of DNA/RNA nanostructures.